人端粒酶蛋白催化亚单位基因逆转录病毒真核表达质粒的构建及其对人乳腺癌相关成纤维细胞增殖的影响

Construction of retroviruses eukaryotic expression vector for human telomerase reverse transcriptase and its effect on proliferation of human breast cancer-associated fibroblasts

目的构建人端粒酶蛋白催化亚单位(Human telomerase reverse transcriptase,hTERT)逆转录病毒真核表达质粒,并检测其在人乳腺癌相关的成纤维细胞(Cancer-associated fibroblast,CAF)中的表达及其对CAF增殖的影响。方法扩增pIRES-EGFP-hTERT质粒,酶切回收hTERT片段,正向克隆至逆转录病毒载体pBABE-puro上,构建重组质粒pBABE-puro-hTERT,转染PT67细胞,进行逆转录病毒的包装。将PT67细胞上清感染原代培养的人乳腺癌CAF,荧光定量PCR和Westernblot分别检测hTERT基因在人乳腺癌CAF中的转录和表达,流式细胞术检测细胞的增殖情况。结果重组质粒pBABE-puro-hTERT经酶切和测序证实构建正确;与未处理的人乳腺癌CAF和感染pBABE-puro的细胞相比,感染重组质粒的人乳腺癌CAF中hTERT基因mRNA的转录水平及蛋白的表达水平均明显增强,其增殖指数也明显增加(P<0.05)。结论成功构建了hTERT基因逆转录病毒真核表达质粒,其能增强人乳腺癌CAF的增殖能力,为乳腺癌微环境CAF的研究提供了良好的细胞模型。

Objective To construct and identify a retroviruses eukaryotic expression vector for human telomerase reverse transcriptase（hTERT）,determine its expression in human breast cancer-associated fibroblasts（CAFs）and observe its effect on proliferation of CAFs.Methods Plasmid pIRES-EGFP-hTERT plasmid was reproduced and digested with enzyme to obtain hTERT gene fragment which was inserted to retroviruses eukaryotic expression vector pBABE-puro of sense orientation.The constructed recombinant plasmid pBABE-puro-hTERT was transfected into PT67 cells for packaging of retrovirus.Primary culture of human breast CAFs were infected with the supernatant of PT67 cells,then determined for transcription and expression of hTERT gene by PCR and Western blot respectively,and for proliferation by flow cytometry.Results Restriction analysis and sequencing proved that recombinant plasmid pBABE-puro-hTERT was constructed correctly.The mRNA transcription and protein expression levels of hTERT as well as the proliferation index of human breast CAFs infected with plasmid pBABE-puro-hTERT increased significantly as compared with those uninfected and those infected with plasmid pBABE-puro.Conclusion A retroviruses eukaryotic expression vector for hTERT was successfully constructed,which enhanced the proliferation of human breast CAFs in vitro.It provided a good cell model for study on microenvironment of human breast CAFs.